Immuno-nephelometry of lipoprotein (a) and reagent therefor

ABSTRACT

Provided is a method for quantitatively measuring an antigen having diverse phenotypes with accuracy in immunoassay. The present invention is particularly intended to latex turbidimetric immunoassay using the antigen-antibody reaction of an antigen having phenotypes, wherein, in detection utilizing the immunoassay, the amount of an antibody against the antigen added to an assay system is adjusted and a basic amino acid is added to the assay system, thereby circumventing the variability of a measurement value attributable to phenotype variety and obtaining a measurement value having a high correlation with a measurement value of the antigen in a biological sample that is measured on a molecular basis.

TECHNICAL FIELD

The present invention relates to a method for quantitatively measuringan antigen having diverse phenotypes with accuracy in immunoassay.

BACKGROUND ART

In immunoassay, the quantitative determination on a molecular basis ofan antigen having diverse phenotypes each differing in its molecularweight has required enzyme-linked immunoassay (ELISA) using severalmonoclonal antibodies that recognize a different antigenic site. Forexample, lipoprotein(a), a protein in blood, has a structure whereapo(a), one of apoproteins, is bound with LDL. Apo(a) has repeats ofdomain structures exhibiting a high homology with plasminogen kringle 4.Because the number of this repeat varies according to an individual, themolecular weight of apo(a) is diversified and Lp(a) (lipoprotein(a)) isallowed to have a variety of phenotypes. For example, Uterrnann, G., et.al. have reported that lipoprotein(a) is broadly divided into 6phenotypes (Utermann G. et al., J Coin Invest 1987; 80: 458-465).Therefore, when a molecule of a phenotype with a certain molecularweight is used as a reference material to measure molecules of otherphenotypes with molecular weights different therefrom in measurement ona weight basis commonly used in immunoassay, a value that deviates froma measurement value measured on a molecular basis is obtained because ofdifference in the molecular weight of each phenotype. In this case,there is also a problem that phenotypic differences result in differencein reactivity with a certain antibody. For quantitatively measuringmolecules of all phenotypes with accuracy regardless of phenotypicdifferences, ideal is not measurement on a weight concentration basisgenerally used for measuring proteins in serum but measurement on amolecular basis by molar concentration. Theoretically, enzyme-linkedimmunoassay using a monoclonal antibody that reacts in a one-to-onerelationship with an antigen is desirable for performing measurement ona molecular basis using immunoassay, and the use of several monoclonalantibodies is required for dealing with diverse phenotypes in themeasurement (Clin Chem 1995; 41: 245-255). On the other hand, in themeasurement of lipoprotein(a), turbidimetric immunoassay using apolyclonal antibody is also widely used, which generally aims atmeasurement on a weight concentration basis. Therefore, theturbidimetric immunoassay has presented a problem of a measurement valuethat deviates from those obtained by enzyme-linked immunoassay due tothe phenotypic differences of an antigen, (Curr Cardiol Rep 1999; 1:105-111).

Lipoprotein(a) is associated with arteriosclerosis, ischemic heartdisease, and so on. The concentration of lipoprotein(a) in blood can beused in the assessment of morbidity risk for these diseases. Under suchcircumstances, a method capable of rapidly and conveniently measuringthe accurate concentration of lipoprotein has been desired.

DISCLOSURE OF THE INVENTION

An object of the present invention is to solve a problem of ameasurement value obtained by turbidimetric immunoassay that deviatesfrom those obtained by enzyme-linked immunoassay due to the phenotypicdifferences of an antigen. Specifically, the present invention isintended to provide a method of bringing a measurement value obtained byturbidimetric immunoassay into agreement with those obtained byenzyme-linked immunoassay by the adjustment of reagent components.

The present inventors have found that the adjustment of reagentcomponents allows the control of a measurement value in turbidimetricimmunoassay for measuring an antigen having several phenotypes. Namely,the present inventors have completed the present invention by findingthat a large amount of an antibody and a given amount of a basic aminoacid such as arginine are added to reagent components, therebycircumventing the influence of variations in measurement valuesattributable to phenotypic differences and obtaining a measurement valuehaving a high correlation with a measurement value obtained byenzyme-linked inmunoassay that is capable of measurement on a molecularbasis.

That is, the present invention is as follows:

-   [1] Latex turbidimetric immunoassay using an antigen-antibody    reaction of lipoprotein(a) having several phenotypes, wherein, in    detection utilizing the immunoassay, the amount of an antibody    against the lipoprotein(a) added to an assay system is adjusted and    a basic amino acid is added to the assay system, thereby    circumventing the variability of a measurement value attributable to    phenotype variety and obtaining a measurement value having a high    correlation with a measurement value of the lipoprotein(a) in a    biological sample that is measured on a molecular basis;-   [2] The immunoassay of [1], wherein the amount of the antibody added    is greater than or equal to 0.16 mg/mL in a reaction solution at the    time of the antigen-antibody reaction;-   [3] The immunoassay of [2], wherein the amount of the antibody added    is from 0.16 mg/mL to 0.23 mg/mL inclusive in the reaction solution    at the time of the antigen-antibody reaction;-   [4] The immunoassay of any of [1] to [3], wherein the amount of the    basic amino acid added is greater than or equal to 15% by weight in    the reaction solution at the time of the antigen-antibody reaction;-   [5] The immunoassay of [4], wherein the amount of the basic amino    acid added is from 15% by weight to 17% by weight inclusive in the    reaction solution at the time of the antigen-antibody reaction;-   [6] The immunoassay of any of [1] to [5], wherein the basic amino    acid is arginine;-   [7] A detection reagent for latex turbidimetric immunoassay using an    antigen-antibody reaction of lipoprotein(a) having phenotypes, the    reagent comprising: an antibody against the lipoprotein(a) in such    an amount that the amount of the antibody is greater than or equal    to 0.16 mg/mL in a reaction solution at the time of the    antigen-antibody reaction; and a basic amino acid in such an amount    that the amount of the basic amino acid is greater than or equal to    15% by weight in the reaction solution at the time of the    antigen-antibody reaction, wherein the latex turbidimetric    immunoassay circumvents the variability of a measurement value    attributable to phenotype variety and obtains a measurement value    having a high correlation with a measurement value of the    lipoprotein(a) in a biological sample that is measured on a    molecular basis;-   [8] The detection reagent for latex turbidimetric immunoassay of    [7], wherein the amount of the antibody added is from 0.16 mg/mL to    0.23 mg/mL inclusive in the reaction solution at the time of the    antigen-antibody reaction;-   [9] The detection reagent for latex turbidimetric immunoassay of [7]    or [8], wherein the amount of the basic amino acid added is from 15%    by weight to 17% by weight inclusive in the reaction solution at the    time of the antigen-antibody reaction; and-   [10] The detection reagent for latex turbidimetric immunoassay of    any of [7] to [9], wherein the basic amino acid is arginine.

The present specification encompasses contents described in thespecifications and/or drawings of Japanese Patent Application No.2003-094059 that serves as a basis for the priority of the presentapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the correlation between measurement valuesobtained by control assay and measurement values obtained by a latexagglutination method in each concentration of an antibody;

FIG. 2 is a diagram showing the deviation of measurement values ofsamples of each phenotype from a regression line in each concentrationof an antibody;

FIG. 3 is a diagram showing the correlation between measurement valuesobtained by control assay and measurement values obtained by a latexagglutination method in each concentration of arginine; and

FIG. 4 is a diagram showing the deviation of measurement values ofsamples of each phenotype from a regression line in each concentrationof arginine.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The present invention is intended to a method of circumventing thevariability of a measurement value attributable to the phenotypicdifferences of an antigen in the detection, using turbidimetricimmunoassay, of the antigen having diverse phenotypes. The antigen to bedetected is not limited as long as it is a protein having severalphenotypes. A protein having a varying molecular weight attributable tophenotypic differences is preferable, with lipoprotein(a) particularlypreferred. It is known that lipoprotein(a) has 6 phenotypes designatedas F, B, S1, S2, S3, and S4 (Nippon Rinsho in Japanese (Japanese Journalof Clinical Medicine) 1999; 57 Suppl: 42-44). These phenotypes can bedetermined by the method of Utermann et al. (Utermann, G. et al., Proc.Natl. Acad. Sci. USA 1989; 86: 4171-4174): the phenotypes are classifiedaccording to their relative mobility compared to the mobility of apo-Bon SDS-PAGE, as F (faster, with a molecular weight smaller than that ofapo-B), B (equal), and S1, S2, S3 and S4 (all slower, in descendingorder of mobility).

Turbidimetric immunoassay is a method in which an antibody contained inan assay reagent is subjected to antigen-antibody reaction with anantigen in a biological sample to form an aggregate whose formation ismeasured by absorbance to qualitatively or quantitatively determine theantigen in the biological sample. For example, the antibody used in theturbidimetric immunoassay is sensitized (bound) to an insoluble carrier.No limitation is imposed on the carrier used, and, for example, latexparticles, bentonite, collodion, kaoline, and immobilized sheeperythrocytes can be used. The latex particles are preferred.Turbidimetric immunoassay using latex particles is called latexturbidimetric immunoassay, in which the latex particles sensitized(bound) with an antibody is mixed with an antigen in a biological sampleto be measured to form the agglutination of the latex particles whosedegree is measured by absorbance to qualitatively or quantitativelydetermine the antigen. The biological sample used in the presentinvention is not limited, and blood, serum, and plasma can preferably beused.

Examples of the latex particles include polystyrene latex particles ofhomopolymers and/or copolymers of vinyl monomers such as vinyl chloride,acrylonitrile, vinyl acetate, acrylic ester, and methacrylic ester,butadiene-based copolymer latex particles such as styrene-butadienecopolymers and methyl methacrylate-butadiene copolymers, andpolyvinyltoluene latex particles. Among them, polystyrenic latexparticles are preferred in that they have the excellent property ofadsorbing, for example, a variety of proteins or polypeptides and canstably maintain biological activity for a long period. Theabove-described latex particles have a particle size of preferably 0.01to 1 μm, more preferably 0.1 to 1 μm. The latex particles having aparticle size less than 0.01 μm lead to frequent occurrence of fineagglutination and ununiformity of an apparent particle size andadversely affect simultaneous reproducibility and so on. Moreover, insome cases, these latex particles are not agglutinated sufficiently forthe number of antibodies. If a particle size is larger than 1 μm,autoagglutination is promoted and dispersibility is reduced.

The antibody used in sensitization of the latex particles is preferablya polyclonal antibody from a rabbit, a goat, a sheep, a pig, a horse, orthe like. The polyclonal antibody can be obtained by immunizing ananimal with purified lipoprotein(a) that serves as an immunogen to giveantiserum, from which the polyclonal antibody is then purified by anappropriately selected method or combined methods from methods known inthe art such as ammonium sulfate precipitation, ion exchangechromatography that employs an anion exchanger such as DEAE cellulose,molecular sieve chromatography that conducts separation according tomolecular sizes or shapes, hydroxyapatite chromatography, and affinitychromatography. No limitation is imposed on the phenotype of thelipoprotein(a) used in this immunization. For the lipoprotein(a),fractions with a specific gravity of 1.063 to 1.15 can be obtained from,for example, normal human serum by ultracentrifugation. The fraction canbe purified by chromatography that uses a column of Sepharose CL-4B andso on.

A method of sensitizing the latex particles with the antibody is notparticularly limited. For example, the antibody may be adsorbedphysically or bound chemically to the carrier. To be more specific, thecarrier can be sensitized with the antibody by mixing the antibody withthe carrier, followed by heating and shaking at 30 to 37° C. for 1 or 2hour(s). The amount of the antibody with which the carrier is sensitizedcan appropriately be set according to the particle size of the carrierused. It is preferred that unsensitized sites on the surface of thecarrier should be blocked with bovine serum albumin, human serumalbumin, rabbit serum albumin, ovalbumin, or the like, after the carrieris sensitized with the antibody. It is preferred that the carriersensitized with the polyclonal antibody should be stored as a mediumdispersion until it is reacted with the biological sample. For example,a phosphate buffer, a glycine buffer and the like can be used as amedium for dispersing the carrier. The content of the carrier sensitizedwith the polyclonal antibody can be typically 0.05 to 0.5% by weight,preferably 0.1 to 0.3% by weight, relative to the amount of the mediumdispersion. Bovine serum albumin, gelatin, gum arabic, or the like maybe added to the medium, if necessary.

Latex turbidimetric immunoassay can be conducted by mixingantibody-sensitized latex particles with a biological sample in anappropriate buffer. In the present invention, a larger amount of theantibody and a basic amino acid is allowed to exist in a reaction systemwhen the antibody-sensitized latex particles are mixed with thebiological sample and agglutinated. The final concentration of anantibody added to a reaction system is 0.05 to 0.1 mg/mL in typicalturbidimetric inmunoassay, whereas in the present invention, theantibody is added at a concentration greater than or equal to at least0.15 mg/mL, preferably greater than or equal to 0.16 mg. An upper limitthereto is not limited in light of the effect of suppressing thevariability of a measurement value attributable to phenotype variety.However, the concentration is preferably 1 mg/mL or less, morepreferably 0.3 mg/mL or less, particularly preferably 0.23 mg/mL orless, from the viewpoint of obtaining a favorable image of agglutinationin the latex turbidimetric immunoassay. For adjusting the amount of theantibody added, the amount of the antibody-sensitized latex particlesadded to the reaction system may be increased, or the amount, per unitlatex particle, of the antibody used for sensitization of the latexparticles may be increased. Alternatively, the antibody-sensitized latexparticles, which are prepared as sensitized latex particles at aconcentration of 0.05 to 0.5% by weight relative to the mediumdispersion as described above, may be allowed to have a higherconcentration in the medium dispersion.

Examples of the basic amino acid include arginine, histidine, glutamine,asparagine, and citrulline. Among them, arginine is particularlypreferred. The amount of the basic amino acid added is greater than orequal to 12%, preferably greater than or equal to 15%, in terms of itsfinal concentration in the reaction system when the antibody-sensitizedlatex particles are mixed with the biological sample and agglutinated.An upper limit of the concentration of the basic amino acid is notlimited in light of the effect of suppressing the variability of ameasurement value attributable to phenotype variety. For example, thebasic amino acid can be used at a concentration on the order of 40%.However, the concentration is preferably 25% or less, more preferably20% or less, particularly preferably 17% or less, from the viewpoint ofobtaining a favorable image of agglutination, although arginine.

A latex agglutination method according to the present invention istypically performed by mixing 1 volume of a biological sample with 40volumes of a first reagent containing a buffer and 20 volumes of asensitized latex particle solution (second reagent). However, any ratioat which abundance ratios of an antigen in the biological sample and asensitized latex particle are proper and a favorable image ofagglutination is obtained may be used without being limited to thismixing ratio. Alternatively, the first reagent and the second reagentmay be used in a single solution. The concentration of the particle inthe sensitized latex particle solution may appropriately be adjustedaccording to a ratio of the volume of the sensitized latex particlesolution added to a reaction system to the total volume of the reactionsystem. For example, a phosphate buffer, pH 7, or glycine buffer, pH 7,are used as the buffer constituting the first reagent. A basic aminoacid may be added to the first reagent, the second reagent, or both, aslong as its final concentration falls in the above-describedconcentration range. In the typical example described above, thepreferred concentration of the antibody in the sensitized latex particlesolution is from 0.5 mg/mL to 0.7 mg/mL inclusive. For example, when thebasic amino acid is added to both of the first reagent and the secondreagent, the basic amino acid may be added at a concentration of 7 to10% for the first reagent and 30% for the second reagent. The preferredconcentration of the antibody or the basic amino acid in these reagentscan readily be determined from the final concentration in the finalreaction system and the volume ratio of each of the reagents.

Reaction is performed by mixing the biological sample with the firstreagent and the second reagent. Although the order of mixing is notlimited, the biological sample may be mixed initially with the firstreagent with stirring for a several minutes, preferably 1 to 5minute(s), more preferably 5 minutes, and subsequently mixed with thesecond reagent with stirring. Agglutination reaction is performed for 1to 4 minute(s) to examine the degree of agglutination. A temperature atwhich the above-described procedures are performed is not limited, butpreferably 37° C. These procedures can also be performed within aplastic cell or a glass cell. In this case, the cell is irradiated fromthe outside with light from visible light to light in the near-infraredregion, for example, a light having a wavelength of usually 400 to 2400nm, preferably 550 to 800 nm. A change in absorbance or a change inscattering light intensity is then detected to measure the degree ofagglutination of the carrier particles. In this case, the use of acalibration curve prepared in advance allows the calculation of theamount (concentration) of the lipoprotein(a) in the sample. Thephenotype of the lipoprotein(a) that is used for preparing thiscalibration curve is not limited. The agglutination can be measuredusing, for example, a fully-automated latex agglutination measurementapparatus LPIA-S500 (Mitsubishi Chemical), an automated analyzerTBA-200FR (Toshiba), and a Hitachi 7170 automated analyzer (Hitachi).

The agglutination reaction may be conducted in a solution such as aphysiological salt solution or an appropriate buffer (pH 5.0 to 10), forexample, a phosphate buffer, a borate buffer, a Tris buffer and thelike.

According to the method of the present invention, the variability of ameasurement value attributable to phenotype variety is circumvented, anda measurement value having a high correlation with a measurement valueof lipoprotein(a) in a biological sample that is measured on a molecularbasis is obtained. The “measurement value of lipoprotein(a) in abiological sample that is measured on a molecular basis” used hereinrefers to, for example, a value measured by enzyme-linked immunoassay(ELISA) conducted under particular conditions.

The ELISA (Enzyme Linked Immuno-Sorbent Assay) method is a method inwhich an antibody is labeled with an enzyme and a substance (antigen)that binds to the antibody is detected. Especially, the ELISA method iswidely used as a method of detecting an antigen protein and as ananalysis method of using antigen-antibody reaction to detect an antigenprotein in a sample or conversely, an antibody that binds to aparticular antigen protein. The ELISA method detects an antibody reactedwith an antigen to be measured, using a second antibody chemically boundin advance with an enzyme such as peroxidase or galactosidase, anddetects the presence or absence or the amount of antigen of interest onthe basis of the degree of color developed by adding, to a reactionsystem, a substrate that develops a color through enzyme reaction.

The general mainstream of the detection of a protein of interest in theELISA method is detection by an antibody-based sandwich method in whichan antigen is sandwiched between an immobilized antibody and a labeledantibody using the combination of a polyclonal antibody and a polyclonalantibody, a monoclonal antibody and a monoclonal antibody, or apolyclonal antibody and a monoclonal antibody (“Seikagaku Jikkenho inJapanese (Experiments in Biochemistry) 11—Immunoassay,” published byTokyo Kagaku Dozin, Nov. 15, 1989).

In the ELISA method, the binding of one molecule of the immobilizedantibody and one molecule of the antigen-one molecule of the labeledantibody is generally established. Unless several epitopes (antigenicdeterminants) recognized by the labeled antibody are present on oneantigen molecule, the above-described binding is established even forany phenotype of an antigen having diverse phenotypes, so thatmeasurement on a molecular basis is made possible. That is, even when amolecule of any phenotype is used as a reference material, molecules ofthe other phenotypes can accurately be measured on a molecular basis.

For example, in lipoprotein(a), a molecule of apo(a), one of structuralproteins, has repeats of structures called kringle 4, which are dividedinto 10 different types from type 1 to type 10. Of these types, kringle4 type 1 and type 3 to type 10 are present as a single copy in allapo(a) species, whereas kringle 4 type 2 is present in a variable numberof repeats in each apo(a) molecule, allegedly varying from 3 to 40.Namely, variations in a molecular weight attributable to the phenotypicdifferences of lipoprotein mainly result from the number of kiingle 4type 2 repeats. This indicates that accurate measurement on a molecularbasis can not be conducted in the measurement using ELISA, oflipoprotein(a) from the number of the repeat of each kringle 4 type, ifan antibody against kringle 4 type 2 is contained in both immobilizedand labeled antibodies (Clin Chem 1995; 41: 245-255). Moreover,identical or similar epitopes are present on domains of differentkringle 4 types. This rather allows measurement on a molecular basis,because the binding of one molecule of the immobilized antibody-onemolecule of the antigen-one molecule of the labeled antibody isestablished without exception when the immobilized antibody and thelabeled antibody are selected from among antibodies capable of bindingto any of kringle 4 type 1 and kringle 4 type 3 to type 10 andrecognizing an epitope that is not commonly present in different kringle4 types and the immobilized antibody and the labeled antibody bind to aseparate kringle 4 type. An antibody that recognizes an epitope presentin a domain other than the kringle 4 domain and present in a single copyin apo(a) may be used as at least the labeled antibody, preferably bothof the immobilized antibody and the labeled antibody. In the presentinvention, the “measurement value of lipoprotein(a) that is measured ona molecular basis” refers to a measurement value obtained by ELISAconducted under such a condition that the binding of one molecule of theimmobilized antibody-one molecule of the antigen-one molecule of thelabeled antibody is established. Examples of such ELISA include ELISAdescribed in Clin Chem 1995; 41: 246-255. When a measurement valueobtained by such an ELISA method is correlated with the measurementvalue obtained by the method of the present invention, its correlationcoefficient (R²) is 0.97 or more, preferably 0.98 or more, still morepreferably 0.99 or more.

The turbidimetric immunoassay of the present invention that circumventsthe variability of a measurement value attributable to phenotype varietyand obtains a measurement value having a high correlation with ameasurement value of lipoprotein(a) in a biological sample that ismeasured on a molecular basis is turbidimetric immunoassay in which acalibration curve prepared by the same measurement as below -using anylipoprotein(a) phenotype as sample substantially shows parallelism witha calibration curve prepared by plotting the relationship of measurementvalues expressed as theoretical concentrations and absorbance variationsin the measurement by the turbidimetric immunoassay, of a preparationobtained by using a lipoprotein(a) reference material (PRM; IFCCproposed reference material) certified by IFCC (International Federationof Clinical Chemistry and Laboratory Medicine, Via Carlo Farini 81,20159 Milano, Italy) as a sample that is in turn subjected to serialdilution with a physiological saline.

The method of the present invention circumvents the variability of ameasurement value attributable to phenotype variety and obtains ameasurement value having a high correlation with a measurement value oflipoprotein(a) in a biological sample that is measured on a molecularbasis. This means that a nearly identical measurement value is obtainedat all times even if the phenotype of a molecule used as a referencematerial is changed. Thus, the method of the present invention is amethod that obtains a constant measurement value, independently of thephenotype of a molecule used as a reference material.

Hereinafter, the present invention will be described specifically withreference to Examples. However, the present invention is not intended tobe limited to Examples below.

EXAMPLE 1

Change in Measurement Value Obtained using Reagents having VaryingConcentrations of Latex (Amounts of Antibody) (Correlation with ControlMethod (ELISA))

A rabbit anti-human lipoprotein(a) polyclonal antibody (available fromDAKO) was mixed with latex particles (available from Sekisui Chemical)and heated at room temperature for 60 minutes and subsequently in athermostat bath at 60° C. for 50 minutes, followed by cooling in coldwater for 20 minutes to thereby conduct sensitization. The polyclonalantibody was sensitized in an amount of 0.14 mg per mg of the latexparticles. The sensitized latex particles were dispersed at aconcentration of 0.5% by weight in 0.17 M glycine buffer, pH 7, to givea rabbit anti-human lipoprotein polyclonal antibody dispersion.

Dispersed suspensions of the latex particles sensitized with the rabbitanti-human lipoprotein(a) polyclonal antibody were prepared so that thefinal concentrations of the antibody in second reagents were brought toapproximately 0.3, 0.4, 0.5, and 0.7 mg/mL, respectively. The dispersedsuspensions were used as the second reagents. A glycine buffersupplemented with 0.1 M NaCl, 0.05 M EDTA, 1% BSA, and 25 mg/mL normalrabbit globulin for the purpose of preventing nonspecific reaction wasused as a first reagent. Arginine as a basic amino acid was added at aconcentration of 10% for the first reagent and 30% for the secondreagent. The final concentration of the arginine in a reaction solutionwas 16.7%.

Twenty human serum samples respectively containing a differentlipoprotein(a) phenotype were used as samples to be measured. Among 20samples, 4 samples each for each of phenotypes B, S1, S3, S4, and S5were used (available from Technoclone).

For performing reaction, 160 μL of the first reagent is added to 4 μL ofeach serum sample and stirred. After five minutes, 80 μL of each secondreagent was added and stirred to conduct agglutination reaction. AHitachi 7170 automated analyzer was used in measurement and set tomeasure the agglutination reaction at 37° C. for 4 minutes from about 1minute into the reaction as absorbance variations at a wavelength of 570nm. For calculating concentrations from the absorbance variations, acalibration curve showing the relationship between the concentration andthe absorbance variations was prepared in advance by using a referencematerial (available from Technoclone) having a known concentration as asample and measuring it under the same condition.

The above-described 20 samples were measured as follows by enzyme-linkedimmunoassay that serves as a control method. A monoclonal antibody usedcan be obtained by a method described in Clin Chem 1995; 41: 246-255. Ananti-human lipoprotein(a) monoclonal antibody (which binds to Kringle 4type 2 but does not bind to kringle 4 type 1 and type 3 to type 10) wasimmobilized at 0.5 μg/well in 96-well microtiter plate manufactured byNunc (100 μL of 5 μg/mL antibody solution in 0.1 M sodium bicarbonatebuffer (pH 9.6) was placed in the well and stirred at room temperaturefor 1 hour, followed by overnight incubation at 4° C.). The wells werewashed three times with PBS, pH 7.4. The plate was blocked by theaddition of 300 μL of PBS containing 30 g/L BSA to the wells andone-hour incubation at room temperature. Following blocking, the wellswere washed three times with PBS, pH 7.4. Then, 100 μL of each of theabove-described samples was added to the wells and stirred at 28° C. for1 hour. Before the addition, the samples were appropriately diluted withPBS containing 1 g/L BSA and 0.5 mL/L Tween 20. After the wells werewashed three times with PBS, pH 7.4, 100 μL of a HRP-labeled anti-humanlipoprotein(a) monoclonal antibody (which binds to a kringle 4 domainother than kringle 4 type 2 domain) solution was added and stirred at28° C. for 1 hour. Subsequently, color reaction was performed by addingOPD and H₂O₂. After 15 minutes, the reaction was stopped by adding 100μL of 1 mol/L sulfuric acid. The absorbance of the reaction solutionsafter the termination of the reaction was measured at 495 nm. Forcalculating concentrations from the absorbance variations, a calibrationcurve showing the relationship between the concentration and theabsorbance variations was prepared in advance by using a referencematerial having a known concentration as a sample and measuring it underthe same condition.

As a result, the samples having measurement values that deviate from aregression line determined by a correlation with enzyme-linkedimmunoassay were observed in the measurement with the reagents where theconcentrations of the antibody in the second reagents were 0.3 mg/mL and0.4 mg/mL, respectively, whereas no sample having a measurement valuethat deviate from the regression line determined by a correlation withenzyme-linked immunoassay was observed in the measurement with thereagents where the concentrations of the antibody in the second reagentswere 0.5 mg/mL and 0.6 mg/mL, respectively. FIG. 1 shows the correlationbetween the measurement values obtained by the control assay and themeasurement values obtained by the latex agglutination method in eachconcentration of the antibody. FIG. 2 shows the deviation of themeasurement values of the samples of each phenotype from the regressionline.

It has been confirmed the adjustment of the concentration of an antibodyin a reagent allows agreement with a measurement value obtained byenzyme-linked immunoassay that serves as a control.

EXAMPLE 2

Change in Measurement Value Obtained using Reagents having VaryingConcentrations of Arginine (Correlation with Control Method (ELISA))

A dispersed suspension of latex particles sensitized with a rabbitanti-human lipoprotein(a) polyclonal antibody was prepared in the sameway as Example 1.

The dispersed suspension of the latex particles sensitized with therabbit anti-human lipoprotein(a) polyclonal antibody was prepared sothat the final concentration of the antibody in a second reagent wasbrought to approximately 0.7 mg/mL. Arginine as a basic amino acid wasadded to the first reagent so that the final concentrations of argininein reaction solutions were brought to 10%, 15, and 17%, respectively(the concentration in the second reagent is 30%).

Measurement conditions and samples to be measured were the same asExample 1.

As a result, the samples having measurement values that deviate from aregression line determined by a correlation with enzyme-linkedimmunoassay were observed when the reaction solution having theconcentration of arginine of 10% was used, whereas no sample having ameasurement value that deviate from the regression line determined by acorrelation with enzyme-linked inmunoassay was observed when thereaction solutions having the concentrations of arginine of 15% and 17%,respectively, were used. FIG. 1 shows the correlation between themeasurement values obtained by the control assay and the measurementvalues obtained by the latex agglutination method in each concentrationof the arginine. FIG. 2 shows the deviation of the measurement values ofthe samples of each phenotype from the regression line.

It has been confirmed the adjustment of the concentration of arginine ina reagent allows agreement with a measurement value obtained byenzyme-linked immunoassay.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

As described in Examples, latex turbidimetric immunoassay can circumventthe variability of a measurement value attributable to the phenotypicdifferences of human lipoprotein(a) having diverse phenotypes byincreasing the amount of an antibody added to a reaction system andadding a basic amino acid having a given concentration to the reactionsystem.

The present invention allows rapid and convenient measurement with anautomated analyzer, which gives a measurement value having an excellentcorrelation with a measurement value obtained by enzyme-linkedimmunoassay, when an antigen having diverse phenotypes is measured.

1. Latex turbidimetric immunoassay using an antigen-antibody reaction oflipoprotein(a) having several phenotypes, wherein, in detectionutilizing the immunoassay, the amount of an antibody against thelipoprotein(a) added to an assay system is adjusted and a basic aminoacid is added to the assay system, thereby circumventing the variabilityof a measurement value attributable to phenotype variety and obtaining ameasurement value having a high correlation with a measurement value ofthe lipoprotein(a) in a biological sample that is measured on amolecular basis.
 2. The immunoassay according to claim 1, wherein theamount of the antibody added is greater than or equal to 0.16 mg/mL in areaction solution at the time of the antigen-antibody reaction.
 3. Theimmunoassay according to claim 2, wherein the amount of the antibodyadded is from 0.16 mg/mL to 0.23 mg/mL inclusive in the reactionsolution at the time of the antigen-antibody reaction.
 4. Theimmunoassay according to any one of claims 1 to 3, wherein the amount ofthe basic amino acid added is greater than or equal to 15% by weight inthe reaction solution at the time of the antigen-antibody reaction. 5.The immunoassay according to claim 4, wherein the amount of the basicamino acid added is from 15% by weight to 17% by weight inclusive in thereaction solution at the time of the antigen-antibody reaction.
 6. Theimmunoassay according to any one of claims 1 to 5, wherein the basicamino acid is arginine.
 7. A detection reagent for latex turbidimetricimmunoassay using an antigen-antibody reaction of lipoprotein(a) havingphenotypes, the reagent comprising: an antibody against thelipoprotein(a) in such an amount that the amount of the antibody isgreater than or equal to 0.16 mg/mL in a reaction solution at the timeof the antigen-antibody reaction; and a basic amino acid in such anamount that the amount of the basic amino acid is greater than or equalto 15% by weight in the reaction solution at the time of theantigen-antibody reaction, wherein the Latex turbidimetric immunoassaycircumvents the variability of a measurement value attributable tophenotype variety and obtains a measurement value having a highcorrelation with a measurement value of the lipoprotein(a) in abiological sample that is measured on a molecular basis.
 8. Thedetection reagent for latex turbidimetric immunoassay according to claim7, wherein the amount of the antibody added is from 0.16 mg/mL to 0.23mg/mL inclusive in the reaction solution at the time of theantigen-antibody reaction.
 9. The detection reagent for Latexturbidimetric immunoassay according to claim 7 or 8, wherein the amountof the basic amino acid added is from 15% by weight to 17% by weightinclusive in the reaction solution at the time of the antigen-antibodyreaction.
 10. The detection reagent for latex turbidimetric immunoassayaccording to any one of claims 7 to 9, wherein the basic amino acid isarginine.